The present invention generally relates to an in vitro method for identifying proteins which, in vivo, are covalently post-translationally modified, and for obtaining tranformants encoding such proteins. More particularly, the invention relates to a novel expression library screen for recovering and identifying isoprenylated proteins.
By way of further background, isoprenylated proteins are involved in eukaryotic cell growth and signal transduction. The protein determinant for prenylation is a short carboxy-terminal motif containing a cysteine, to which the isoprenoid is covalently attached via thioether linkage. To date, isoprenylated proteins have almost all been identified by demonstrating the attachment of an isoprenoid to previously known proteins. Thus, many isoprenylated proteins likely remain undiscovered.
Specific prenyl:protein transferases catalyze the transfer of farnesyl (FTase) or geranylgeranyl (GGTase) moieties from isoprenyl pyrophosphates to proteins. The protein determinant for prenylation is a short carboxy-terminal motif containing a cysteine, to which the isoprenoid is covalently attached via thioether linkage. For farnesylation, this motif is CaaX, where `C` is cysteine, `a` is usually an aliphatic amino acid, and `X` is methionine, glutamine, serine, cysteine, or alanine (1). The type I GGTase recognizes a similar CaaX motif with a C-terminal leucine, whereas GGTase II prenylates proteins ending in XXCC, CCXX, or CXC (1). Many signal-transducing fungal and animal proteins are prenylated, including Ras (2-4), Ras-like small GTP-binding proteins (4), receptor kinases (5, 6), heterotrimeric G-protein gamma subunits (7-10) and retinal cGMP phosphodiesterase subunits (11). The biological significance of protein isoprenylation is underscored by the fact that unfarnesylated K-Ra protein is localized to the cytoplasm rather than the plasma membrane, and is incapable of cellular transformation (3, 4). Plants employ signalling pathways similar to those involving prenylated proteins in other organisms (12-14), but analogous prenylated proteins have not been characterized in plants. Nuclear lamins, which form the nuclear lamina and associate with chromatin and the nuclear envelope, are also prenylated in animals (15, 16). Lamin-like proteins have recently been discovered in plant nuclei, but it is not clear if these proteins are prenylated or associated with the nuclear envelope (17-19).
In tobacco suspension cultures, multiple proteins are prenylated in vivo, although their identities are not known (20). One plant protein is known to be prenylated in vivo: a farnesylated Atriplex protein designated ANJ1 that is homologous to the bacterial molecular chaperone DnaJ (21, 22). However, several plant cDNA-encoded small GTP-binding proteins have been shown to contain consensus geranylgeranylation sequences at the carboxy-terminus (23-25), indicating probable prenylation in vivo. Identification of isoprenylated proteins would provide a basis for future work on the role of protein isoprenylation in cellular processes.
A hamster isoprenylated protein was recently identified by purification from .sup.3 H!-mevalonate-labelled cells (26). This approach cannot be readily applied to most eukaryotic tissues, due to insufficient mevalonate incorporation (20).
In light of this background, there persists a need for a method which can be used to readily obtain and identify isoprenylated proteins, both currently known and yet unknown. There also persists a need for methods for identifying transformant microorganisms containing genes encoding such proteins, and for raising antibodies to such proteins. The present invention addresses these needs.